Continuous in- vitro evolution

A method for producing and selecting a mutant protein of interest, the method comprising:

• (a) incubating a replicable RNA molecule encoding the protein with ribonucleoside triphosphate precursors of RNA and an RNA-directed RNA polymerase, wherein the RNA-directed RNA polymerase replicates the RNA molecule but introduces mutations thereby generating a population of mutant RNA molecules;
• (b) translating the mutant RNA molecules in cells wherein, after translation, each mutant protein is associated with or located within the same cell as its encoding RNA molecule;
• and
• (c) selecting a cell comprising a mutant protein of interest.

A method for producing and selecting a mutant protein of interest, the method comprising:

• (a) transcribing a DNA template to produce a replicable RNA molecule, wherein the DNA template comprises:
• (i) an untranslated region comprising a control element that promotes transcription of DNA into RNA and a ribosome binding site;
• (ii) an open reading frame encoding a protein;
• and
• (iii) a stemloop structure situated upstream of the open reading frame;
• (b) incubating the replicable RNA molecule encoding the protein with ribonucleoside triphosphate precursors of RNA and an RNA-directed RNA polymerase, wherein the RNA-directed RNA polymerase replicates the RNA molecule but introduces mutations, thereby generating a population of mutant RNA molecules;
• (c) incubating the mutant RNA molecules with a translation system under conditions which result in the synthesis of a population of mutant proteins;
• (d) selecting one or more mutant proteins of interest.